Many diseases and disorders are characterized by the initiation of biochemical cascades that can result in the injury of cells, tissues and organs. There are two major pathways that can cause damage: hemolytic or ischemic. Within these pathways are complex interactions that can result in metabolic, inflammatory and physiological aberrations. While the hemolytic and ischemic pathways are unique in their initiating factors, they share similar events in their pathogenesis such as inflammation, vasoconstriction and the production of reactive oxygen species. Uncontrolled, these cascades can lead to the development of severe complications which are secondary to the initial insult.
Hemolytic Cascade
Hemolysis is the destruction of red blood cells (RBC) with the subsequent release of hemoglobin (Hb). While hemolysis is part of the normal cycle of RBCs, in certain disorders acellular Hb is released in such large quantities that the normal clearance processes are overwhelmed. Examples of initiating diseases include Sickle Cell Anemia (Sickle Cell Disease or “SCD”), cerebral hemorrhage caused by hemorrhagic stroke or traumatic brain injury, autoimmune hemolytic anemia and other inherited or acquired causes.
Acellular oxygenated Hb (oxyHb) is highly toxic due to its effect on nitric oxide (NO) levels. NO is a mediator of vascular homeostasis that is synthesized in the endothelium and is continually released to regulate vascular tone, and also impacts vascular permeability, apoptosis, platelet adhesion, and leukocyte recruitment and adhesion. It is an integral factor in maintaining vascular homeostasis. OxyHb binds strongly to NO when freed from erythrocytes. Thus, in the presence of acellular oxyHb, NO is readily scavenged and regulation of vascular tone is compromised, inducing vasoconstriction and ischemia. The most profound effects of vasoconstriction are seen at the microvasculature level where vasoconstriction lowers capillary pressure and decreases functional capillary density. This results in poor tissue oxygenation and the accumulation of metabolic by-products that cause microvascular dysfunction and the development of hypoxia (reduced oxygen availability) which initiates the ischemic cascade.
Low-level inflammation in the vascular endothelium is also associated with decreased production of NO. When NO is scavenged by acellular oxyHb, a pro-inflammatory environment is created. Inflammatory pathways are activated that recruit leukocytes, neutrophils and activated microglia cells. Inflammation induced by subarachnoid hemorrhage (SAH) causes brain edema which leads to the development of early brain injury and delayed brain ischemia.
Under the proper redox conditions, NO can be metabolized to a reactive form, peroxynitrite, which can act with other reactive oxygen species (ROS) to interfere with NO synthesis, promote inflammation, cause the dysfunction of critical cellular processes, disruption of cell signaling pathways, and the induction of cell death through both apoptosis and necrosis.
Ischemic Cascade
Ischemia is caused by a reduced blood flow, typically a consequence of a mechanical obstruction in a blood vessel. A number of disease conditions such as atherosclerosis, SCD, colitis, and diabetes can cause blockage of the vasculature leading to reduced blood flow and the development of oxygen deprivation (hypoxia).
Hypoxia rapidly leads to the disruption of mitochondrial function and membrane ion transport. This in turn causes cellular edema and eventually apoptosis or necrosis. Like hemolysis, hypoxia also initiates a cascade which causes damage beyond the site of the initial insult. If unchecked, waves of depolarization, induction of inflammation, generation of ROS and other factors become uncontrolled, causing apoptosis and necrosis to spread significantly beyond the initial core of damage. However, if oxygen can be restored during the acute phases of the cascade, disturbances of the mitochondria, membrane transport and edema can be repaired and irreversible injury and necrosis of the surrounding tissue can be prevented.
Anemia
Anemia is usually defined as a decrease in amount of RBCs or the amount of hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen. There are three main types of anemia: (1) due to blood loss, (2) due to decreased red blood cell production, and (3) due to increased red blood cell breakdown. The severity of anemia has a broad range, due to type of the anemia, genetic variation, and available therapy. However, severe anemia with very low hemoglobin levels is commonly treated with red blood cell transfusions. This does not alter the ability of the patient's own blood to carry oxygen, but simply increases the number of red blood cells that carry oxygen. Blood transfusions, especially in a patient with chronic anemia, raises concerns of disease transmission, volume overload, allo-immunization, hemolytic reactions and transfusion-related acute lung injury.
In diseases such as SCD, the RBCs deform upon releasing oxygen to the tissues, and this causes many of the complications associated with the disease. Normal hemoglobin (Hb-A) exists as a single, isolated protein unit in the RBC membrane that maintains the basic disc shape of the RBC, independently of whether or not it is in the oxygenated or deoxygenated state. However, the abnormal hemoglobin present in SCD (Hb-S) acts differently. In the oxygenated state, Hb-S is found as isolated protein units. But when deoxygenated, these molecules form rigid, polymeric chains of Hb (“fibers”). The rigidity of the polymer causes the structural deformation of the RBC, resulting in irregularly shaped cells. When the sickle cells become re-oxygenated, the Hb-S again is found as an isolated protein unit. As the sickle cells flow through the circulatory system, these undergo repeated cycles of polymerization and de-polymerization. This eventually causes damage to the red blood cell membrane, further compromising its structural integrity and ability to transport oxygen. After recurrent episodes of sickling, membrane damage occurs, and the cells become incapable of resuming the normal biconcave shape upon re-oxygenation.
RBCs expressing Hb-S have unique pathological characteristics. These characteristics are the basis of the comorbidities associated with SCD:                Because of the irregular shape, deoxygenated sickle cells have profound vaso-occlusive behavior leading to the development of ischemia and hypoxia.        Sickle RBCs adhere to endothelium because of increased “stickiness” of the cell membrane. Sickle RBC-endothelial interactions have been implicated as a potential initiating mechanism in vaso-occlusion. Patients with more non-deformable RBCs have more ischemic crises. Sickle cells also adhere to macrophages, thus, contributing to erythrophagocytosis and the hemolytic process.        Sickle RBCs are far more fragile than normal RBCs and readily undergo hemolysis, releasing acellular Hb and other products into the circulation and tissues. It is estimated that the lifespan of a sickle RBC is less than 1/10 that of a normal RBC.Pulmonary Indications        
The inadequate diffusion of oxygen from the lungs into the blood results in hypoxia. While the causes of the lung insufficiency in some cases can be potentially treated by antibiotics or anti-inflammatory drugs, there is a lag time before a response is seen. For many pulmonary diseases, there are no therapeutics that address the underlying disease pathology, and supplemental oxygen via a delivery system such as a Venturi mask or nasal cannula is the only option to increasing the amount of oxygen carried by RBCs. Under severe exacerbations of the disease or emergency situations, continuing respiratory decompensation with worsening carbon dioxide retention and hypoxia despite standard treatment are indications for therapy with non-invasive positive pressure ventilation or endotracheal intubation. However, complications with intubation are common, and include aspiration, esophageal intubation, dental injury, and pneumothorax. The most serious complication is ventilator-associated pneumonia, which develops at a rate of approximately 1% per day and has a mortality rate between 20-50%. Therefore, a therapy that avoids or reduces the time of intubation is highly desired.
Use of Hemoglobin Based Oxygen Carriers
Hemoglobin based oxygen carriers (HBOCs) have long been in development as a substitute for blood transfusions. A number of HBOCs have been in clinical development over the last decade. However, serious side effects (vasoconstriction, system hypertension and oxidative-stress-induced tissue toxicity) and lack of efficacy forced the discontinuation of clinical trials and their development. It is believed that the vasoconstriction and accompanying hypertension are due to the scavenging of NO by the acellular Hb of the HBOC. The depletion of NO initiates a cascade of events that can result in disturbances such as vasoconstriction, inflammation and ROS production. The HBOCs in development varied in the degree of vasoconstriction. Polyethylene glycol (PEG) polymerized forms showed minimal amounts of vasoconstrictive activity. The oxidative stress caused by HBOCs is thought to be due to the inappropriate unloading of oxygen, resulting in the production of ROS and overwhelming normal controlling metabolic processes. Not only were these HBOCs toxic, but the scavenging of NO and increased production of ROS exacerbated the inflammatory, hypoxic, vasoconstrictive and hypertensive conditions already suffered by the patient. An authoritative review article, as of 2008, concluded that, “[b]ased on the available data, use of [HBOCs] is associated with a significantly increase risk of death and [myocardial infarction].” See Natanson C. et al., “Cell-Free Hemoglobin-Based Blood Substitutes and Risk of Myocardial Infarction and Death: A Meta-analysis,” JAMA. 2008; 299(19):2304-2312.
HBOCs have been under development for treatment of SCD, either as blood substitutes or as a means of delivering CO to desickle or depolymerize the aberrant Hb-S. However, none of these products were shown to be able to deliver oxygen to RBCs or to reduce or revert the polymerization of the Hb-S molecule.
HBOCs are also under development for indications where either there was anemia due to trauma or surgery (e.g., hemorrhagic anemia) or due to a genetic defect (e.g., sickle cell disease). In the case of a pulmonary disease such as chronic obstructive pulmonary disease, the hypoxia is not due to anemia, but rather to poor lung function. Unfortunately, such HBOCs did not have characteristics that enabled these products to improve diffusion of oxygen into the blood.
Lack of suitable HBOCs has greatly hindered basic research into the physiology of tissue oxygenation and our understanding of the critical mechanisms involved in shock and its ensuing tissue damage.
Thus, it would be desirable to identify novel oxygen and carbon monoxide carrying and delivering molecules that can serve as blood substitutes and/or have therapeutic activity, while avoiding or ameliorating inflammation, vasoconstriction and hypoxia.